A single application of nicotine induces sustained changes in [Ca²⁺]_i along vHipp axons.

<div><p>Spinning disk confocal live Ca²⁺ images from WT vHipp axons were recorded every 10 seconds for 30 min and fluo-4 fluorescence intensities were calculated and quantified as a normalized integrated intensity at each time point. The changes of normalized integrated intensities at one spot of vHipp axons were plotted vs. time. </p> <p><b>A1</b>: Representative plot from a live WT vHipp axon perfused with normal HEPES buffered solution showed that the normal axonal intracellular Ca²⁺ signals oscillated in a random manner with very small amplitude. <b>A2</b>: Representative spinning disk confocal fluo-4 images are indicated on pseudo color scale at different time points (0, 2.1’, 4.5’, 17.5’, 20.5’, and 23’). The square area is the region for which fluo-4 intensities were quantified. Scale bar: 5μm. <b>A3</b>: Representative spinning disk confocal fluo-4 images are indicated on pseudo color scale in 2D manner from the square area (see A2) at different time points.</p> <p><b>B1</b>: Representative plot from a live WT vHipp axon perfused with nicotine (1μM) for 1 min and then with normal HEPES buffered solution without nicotine. The nicotine application induced both a quick and sustained increase of axonal intracellular calcium signals that oscillated in a random manner with large amplitude. <b>B2</b>: Representative spinning disk confocal fluo-4 imagings are indicated on pseudo color scale at different time points after nicotine application (0, 2’, 4’, 17.5’, 22’, and 25’). The square area is the area for which fluo-4 intensities were quantified. Scale bar: 5 μm. <b>B3</b>: Representative spinning disk confocal fluo-4 images are indicated on pseudo color scale in 2D manner from the square area (see B2) at different time point.</p> <p><b>C</b>: After recording of Fluo-4/Ca²⁺fluorescence (<b>c1</b>), the culture of vHipp microslices were fixed, permeabilized, and stained with antibodies recognizing panaxonal marker (green), MAP2 (red),and GFAP (blue). The vHipp projections previously assessed by calcium imaging and relocated post hoc are co-labeled by panaxonal marker (<b>c2</b>), but not by either a dendritic marker MAP2 (<b>c3</b>), or by a glia marker GFAP (<b>c4</b>), scale bar: 5μm. </p>